TY - JOUR
T1 - Towards delineation of a developmental α-importome in the mammalian male germline
AU - Miyamoto, Yoichi
AU - Baker, Mark A
AU - Whiley, Penelope Alexandra Falshaw
AU - Arjomand, Arash
AU - Ludeman, Justin P
AU - Wong, Chin Long
AU - Jans, David A
AU - Loveland, Katherine A L
PY - 2013
Y1 - 2013
N2 - Nucleocytoplasmic transport mediated by importin proteins is central to many developmental processes, such as precisely regulated germ cell differentiation during spermatogenesis. Here we examine for the first time the dynamic association of importins with cargo during two successive spermatogenic stages: meiotic pachytene spermatocytes and haploid round spermatids of the adult rat testis. Immunoprecipitation followed by mass spectrometry yielded the first non-biased identification of proteins selectively interacting with importin alpha2, alpha3 and alpha4 in each of these cell types. Amongst the 22 novel importin binding proteins identified, 11 contain a predicted classical nuclear localization signal (NLS) for importin alpha binding using a new algorithm (Kosugi et al. [22]), although only 6 of these have known nuclear functions. An importin alpha2-immunoprecipitated protein with a key nuclear role in meiosis, structural maintenance of chromosomes 6 (SMC6), contained a predicted bipartite NLS that was shown to be preferentially recognized by importin alpha together with importin beta1. In contrast, the predicted cNLS of synovial sarcoma, X breakpoint 2 interacting protein (SSX2IP) was found not to confer either nuclear accumulation or direct binding to importin alphas, implying that NLS prediction algorithms may identify cryptic importin binding sites or require additional refinement to increase their accuracy. Unbiased identification of importin alpha binding proteins in cellular differentiation represents a powerful tool to help identify the functional roles of importin alphas.
AB - Nucleocytoplasmic transport mediated by importin proteins is central to many developmental processes, such as precisely regulated germ cell differentiation during spermatogenesis. Here we examine for the first time the dynamic association of importins with cargo during two successive spermatogenic stages: meiotic pachytene spermatocytes and haploid round spermatids of the adult rat testis. Immunoprecipitation followed by mass spectrometry yielded the first non-biased identification of proteins selectively interacting with importin alpha2, alpha3 and alpha4 in each of these cell types. Amongst the 22 novel importin binding proteins identified, 11 contain a predicted classical nuclear localization signal (NLS) for importin alpha binding using a new algorithm (Kosugi et al. [22]), although only 6 of these have known nuclear functions. An importin alpha2-immunoprecipitated protein with a key nuclear role in meiosis, structural maintenance of chromosomes 6 (SMC6), contained a predicted bipartite NLS that was shown to be preferentially recognized by importin alpha together with importin beta1. In contrast, the predicted cNLS of synovial sarcoma, X breakpoint 2 interacting protein (SSX2IP) was found not to confer either nuclear accumulation or direct binding to importin alphas, implying that NLS prediction algorithms may identify cryptic importin binding sites or require additional refinement to increase their accuracy. Unbiased identification of importin alpha binding proteins in cellular differentiation represents a powerful tool to help identify the functional roles of importin alphas.
UR - http://www.sciencedirect.com/science/article/pii/S0167488912003266
U2 - 10.1016/j.bbamcr.2012.11.005
DO - 10.1016/j.bbamcr.2012.11.005
M3 - Article
SN - 0167-4889
VL - 1833
SP - 731
EP - 742
JO - BBA Molecular Cell Research
JF - BBA Molecular Cell Research
IS - 3
ER -